Polymerization of nucleic acids using activation by polyphosphorolysis (app) reactions

ABSTRACT

This disclosure relates to methods of performing activation by polyphosphorolysis (APP) reactions using at least one of the polyphosphorylating agents triphosphate, polyphosphate, imidodiphosphate, thiodiphosphate (or μ-monothiopyrophosphate), and related compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/324,676, filed Dec. 13, 2011, now U.S. Pat. No. 8,932,813, and claimsthe benefit of priority under 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application No. 61/422,477, filed 13 Dec. 2010, each of which areherein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to methods for polymerization of nucleic acidsusing activation by polyphosphorolysis (APP) reactions, in the presenceof improved polyphosphorylating agents.

BACKGROUND OF THE DISCLOSURE

This disclosure relates to methods for polymerization of nucleic acids(or nucleotides) using activation by polyphosphorolysis (APP) reactions.Polymerization may be carried out using primers having 3′-ends blockedwith dideoxynucleotides in a reaction mixture containing a balance ofpyrophosphate (PPi) and dNTP. These methods have been previouslydescribed in, for example, U.S. Pat. Nos. 6,534,269 B2, 7,033,763 B2,7,238,480, 7,105,298, 7,504,221, and 7,919,253. Similar methods are alsodescribed in U.S. Pat. No. 7,745,125. These methods have manyapplications, including detection of rare alleles in the presence ofwild type alleles (e.g., one mutant allele in the presence of 10⁶-10⁹wild type alleles). The methods are useful to, for example, detectminimal residual disease-causing agent(s) (e.g., rare remaining cancercells during remission, especially mutations in the p53 gene or othertumor suppressor genes previously identified within the tumors), and/ormeasure mutation load (e.g., the frequency of specific somatic mutationspresent in normal tissues, such as blood or urine). Other applicationsof such methods are known to those of skill in the art.

Those of skill in the art desire improved methods for carrying out suchmethods. It has been surprisingly found that several other compounds arealso capable of deblocking of nucleic acids. Described herein aremethods for performing an APP reaction using various polyphosphorolyzingagents. Advantages of these methods will be apparent from the followingdescription of such improved methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Titration of human DNA using triphosphate-catalyzed APP.

FIG. 2. Comparison of triphosphate-catalyzed APP with SYBR Green I basedassay.

FIGS. 3A, 3B and 3C. Examples of amplification from a plasmid by PowerSYBR: FIG. 3A is an amplification plot, FIG. 3B is a dissociation curve,and FIG. 3C depicts products run on an agarose gel.

FIGS. 4A, 4B, and 4C. Examples of triphosphate-catalyzed APPamplification from plasmid DNA: FIG. 4A is an amplification plot, FIG.4B is a dissociation curve, and FIG. 4C depicts products run on anagarose gel.

FIG. 5. Comparison of triphosphate- and PPi-catalyzed reactions usingPA-10.

FIGS. 6A, 6B, 6C, 6D, 6E and 6F. Exemplary amplification curves andmelting curves of triphosphate- and PPi-catalyzed reactions. FIG. 6A isan amplification plot of triphosphate-catalyzed reactions and FIG. 6B isan amplification plot of PPi-catalyzed reactions. FIG. 6C is a meltingcurve of triphosphate-catalyzed reactions. FIG. 6D, FIG. 6E, and FIG. 6Fare melting curves of PPi-catalyzed reactions.

FIG. 7. Melting curve of PPi-catalyzed reaction with 125 pg human DNAinput.

FIG. 8. Comparison of triphosphate- and PPi-catalyzed reactions usingPA-17.

FIGS. 9A and 9B. FIG. 9A is a kinetic plot of linear amplification. FIG.9B lists relative rates of linear amplifications of three reactionsusing perfectly matched substrates and mismatches substrates.

FIG. 10. Linear extension by Therminator III in the presence of variousconcentrations of TP

FIG. 11. Deblocking and extension of a ddC-blocked primer by RQY usingMn⁺⁺ as the co-factor.

FIGS. 12A and 12B. FIG. 12A is an amplification plot oftetraphosphate-initiated PCR. FIG. 12B depicts the polyphosphatetetraphosphate.

FIGS. 13A and 13B. FIG. 13A depicts IDP-initiated PCR products run on anagarose gel. FIG. 13B depicts the polyphosphate imidodiphosphate (IDP).

FIG. 14. Amplification curves between primers that has 4-(diamond),6-(square), or 9-(triangle) bases overlaps in regular PCR (filled) andin APP PCR (open).

FIG. 15. Amplification curves of by 48 primers alone in regular PCR(filled), and APP PCR (open).

FIGS. 16A, 16B, 16C and 16D. FIG. 16A depicts an amplification plot forpositive digital PCR (dPCR) using APP reactions and FIG. 16B depictstheir dissociation curves. FIG. 16C depicts an amplification plot fornegative dPCR using APP reactions and FIG. 16D depicts theirdissociation curves.

FIGS. 17A, 17B, 17C and 17D. FIG. 17A and FIG. 17C depict amplificationplots for digital PCR using conventional dye-based master mix and FIG.17B and FIG. 17D depict their dissociation curves, respectively.

FIGS. 18A and 18B. FIG. 18A is a plot depicting the distribution of Ct'sof negative reactions and positive reactions in digital PCR using APPand using conventional dye-based master mix. FIG. 18B is a plot toindicate the mean and 1× standard deviation for the data depicted inFIG. 18A.

SUMMARY OF THE DISCLOSURE

Described herein are methods for amplifying a target nucleic acid from atest sample using activation by polyphosphorolysis (APP) reaction. Incertain embodiments, APP is carried out using one or morepolyphosphorolyzing agents. In some embodiments, the one or morepolyphosphorolyzing agents may be represented by Formula I:

Wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. Insome embodiments, the one or more polyphosphorolyzing agents may berepresented by Formula II:

In some embodiments representing compounds of Formula II, n and/or m maybe the same or different. And n and/or m may be, for example, 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 with the proviso that n or m, but not both,may be 0. Thus, if n is 0, then m may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. If m is 0, then n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. And bothn and m may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, nand m are both 1. In some embodiments, the sum of n and m is greaterthan or equal to 2 (e.g., n≧1 and m≧1, n≧2 and m≧0, n≧0 and m≧2). Insome embodiments, such as where (but not limited to) the sum of n+m isgreater than or equal to 2, X may be, for example,

In some embodiments wherein the one or more polyphosphorolyzing agentsare represented by Formula II, such as where (but not limited to) n orm=0, X may be, for example,

In some embodiments, the one or more polyphosphorolyzing agents may be:

Any of the polyphosphorolyzing agents described herein may be combinedwith any other polyphosphorolyzing agents, such as those describedherein. In some embodiments, the one or more polyphosphorolyzing agentsmay be pyrophosphate (PP_(i)) in combination with at least one or moreother polyphosphorolyzing agents. Any of the one or morepolyphosphorolyzing agents may be used in the form of a suitable salt(e.g., sodium). Typically, the reactions described herein furtherinclude an enzyme having polyphosphorolysis activity. Pyrophosphate hasbeen described as a substrate for pyrophosphorolysis reaction in theliterature (Kornberg, 1968), and in the U.S. Pat. Nos. 6,534,269 B2,7,033,763 B2, 7,238,480, 7,105,298, 7,504,221, and 7,919,253, 7,745,1252(all of which are incorporated by reference in their entirety into thisapplication). APP is typically carried out by: (a) annealing to anucleic acid a first oligonucleotide which has a non-extendable 3′ end(“P*”) removable by polyphosphorolysis (i.e., “activatable”); (b)removing the 3′ non-extendable terminus using one or morepolyphosphorolyzing agents and an enzyme having polyphosphorolysisactivity to produce an unblocked oligonucleotide (i.e., “deblocking”);and, (c) extending the unblocked oligonucleotide to produce a desirednucleic acid strand. In some embodiments, methods are disclosed forproducing an unblocked oligonucleotide from a blocked oligonucleotide(e.g., comprising a non-extendable 3′ end removable bypolyphosphorolysis), the method comprising contacting the blockedoligonucleotide (e.g., when hybridized to a target nucleic acid) withone or more polyphosphorolyzing agents. Such methods typically alsoinclude an enzyme having polyphosphorolysis activity. The enzyme havingpolyphosphorolysis activity for use in any of the methods describedherein may be a DNA polymerase that may also be used to extend theunblocked oligonucleotide. In some embodiments, the enzyme may also havereverse transcriptase activity. In some embodiments, the polymerized oramplified nucleic acid is deoxyribonucleic acid (DNA). In someembodiments, the DNA is human DNA, bacteriophage DNA, plasmid DNA orsynthetic DNA. In some embodiments, the method further comprisesdetecting the desired nucleic acid strand.

Variations of these methods, as well as reagents, kits, and devices forcarrying out the same are also provided. Additional embodiments of themethods described herein may be derived from the description providedbelow.

DETAILED DESCRIPTION

Described herein are methods for polymerizing, amplifying, and/orsequencing a target nucleic acid from a test sample using activation bypolyphosphorolysis (“APP”) reaction. Polyphosphorolysis refers to theremoval of a non-extendable nucleotide from a nucleic acid (e.g., anoligonucleotide) in the presence of one or more polyphosphorolyzingagents and an enzyme that exhibits polyphosphorolyzing activity. APP maybe used to polymerize and/or amplify and/or sequence nucleic acidmolecules, including but not limited to ribonucleic acid (e.g., RNA)and/or deoxyribonucleic acid (e.g., DNA), or hybrids thereof. Other usesfor the methods described herein will be readily understood by one ofskill in the art.

In some embodiments, the one or more polyphosphorolyzing agents may berepresented by Formula I:

wherein n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more. Insome embodiments, the one or more polyphosphorolyzing agents may berepresented by Formula II:

In some embodiments representing compounds of Formula II, n and/or m maybe the same or different. And n and/or m may be, for example, 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 with the proviso that n or m, but not both,may be 0. Thus, if n is 0, then m may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. If m is 0, then n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, n and m are both greater than or equal to 1. In someembodiments, n and m are both 1. In some embodiments, the sum of n+m isgreater than or equal to 2 (e.g., n≧1 and m≧1, n≧2 and m≧0, n≧0 andm≧2). In some embodiments, such as where (but not limited to) the sum ofn+m is greater than or equal to 2, X may be, for example,

In some embodiments wherein the one or more polyphosphorolyzing agentsare represented by Formula II, such as where (but not limited to) n orm=0, X may be, for example,

for example:

In some embodiments, the one or more polyphosphorolyzing agents may be:

Any of the polyphosphorolyzing agents described herein may be combinedwith any other polyphosphorolyzing agents. In some embodiments, the oneor more polyphosphorolyzing agents may be pyrophosphate (PP_(i)) incombination with at least one or more other polyphosphorolyzing agents.Any of the one or more polyphosphorolyzing agents may be used in theform of a salt (e.g., sodium). Typically, the reactions described hereinfurther include one or more biocatalysts (e.g., enzyme(s)) havingpolyphosphorolysis activity to generate one or more nucleosidetriphosphates. As shown above, for example, imidodiphosphate (IDP) linksthe phosphate moieties using nitrogen; similar diphosphate compounds maysubstitute sulfur for nitrogen. In some embodiments, a polyphosphate maybe any phosphate ester having two or more phosphate moieties. In someembodiments, a polyphosphate may be any phosphate esters having three ormore phosphate moieties.

An exemplary one or more biocatalyst that may be used in APP is a DNApolymerase that catalyzes polymerization of nucleoside triphosphates andpolyphosphorolysis of duplexes of DNA in the presence of one or morepolyphosphorolyzing agents as described herein. Exemplary DNApolymerases having polyphosphorolysis activity include but are notlimited to thermostable Tfl, Taq, and/or genetically engineered DNApolymerases (e.g., AMPLITAQFS, THERMOSEQUENASE), those having the activesite mutation F667Y or the equivalent of F667Y (e.g., in Tth) whichshows improved affinity for dideoxynucleotide as incoming nucleotide(e.g., smaller K_(m) for ddNTP)), RQ1 as described in U.S. Pat. No.7,422,872 (incorporated by reference in its entirety into thisapplication) and mutants thereof (e.g., RQY in which 669 is substitutedby tyrosine, which may provide for reverse transcription and/or directsequencing of RNA), THERMINATOR I (NEB), THERMINATOR II, THERMINATORIII, and/or THERMINATOR GAMMA (all available from NEB), among others.These and other potentially suitable DNA polymerases may be describedin, for example, U.S. Pub. 2008/0254525A1, U.S. Pub. 2007/0020622A1,U.S. Pub. 2007/0009924A1, U.S. Pat. No. 4,889,818, U.S. Pat. No.4,965,188, U.S. Pat. No. 5,047,342, U.S. Pat. No. 5,079,352, U.S. Pat.No. 5,270,179, U.S. Pat. No. 5,374,553, U.S. Pat. No. 5,436,149, U.S.Pat. No. 5,512,462, U.S. Pat. No. 5,614,365, and/or U.S. Pat. No.6,228,628B1, all of which are hereby incorporated by reference in theirentirety into this application. It has been found that the use of suchgenetically engineered DNA polymerases may improve the efficiency ofAPP.

APP provides for the extension of oligonucleotides by converting anon-extendable oligonucleotide into an extendable oligonucleotide,extending the oligonucleotide to produce a desired nucleic acid strand(e.g., a complementary copy of a target nucleic acid), and optionallyamplifying and detecting the desired nucleic acid strand. Anon-extendable nucleotide refers to a nucleotide, which uponincorporation into a nucleic acid prevents further extension of thenucleic acid, e.g., by at least one biocatalyst (e.g., enzyme). Anucleotide may be extendable by one enzyme, but non-extendable byanother enzyme. A non-extendable nucleotide to one enzyme could becomeextendable or partially extendable under different conditions. Anextendable nucleotide may refer to a nucleotide to which at least oneother nucleotide can be added or covalently bonded at a 3′-position ofthe sugar moiety of the extendable nucleotide by a biocatalyst (e.g.,enzyme) present in the reaction. Extension may also start from 2′-OH ofa nucleotide which may or may not have an extendable 3′-OH. Extending anucleic acid refers to the addition of or incorporation of one or morenucleotides to or into a given nucleic acid. An extended oligonucleotideis typically an oligonucleotide (e.g., a primer nucleic acid) to whichone or more additional nucleotides have been added or otherwiseincorporated (e.g., covalently bonded to). APP is typically carried outusing the steps of: (a) annealing to a nucleic acid a firstoligonucleotide which has a non-extendable 3′ end (“P*”) that isremovable by polyphosphorolysis (i.e., activatable); (b) removing that3′ non-extendable terminus using a polyphosphorolyzing agent and abiocatalyst (i.e., a DNA polymerase) having polyphosphorolysis activityto produce an unblocked oligonucleotide; and, (c) extending theunblocked oligonucleotide to produce a desired nucleic acid strand.Further steps of detecting the desired nucleic acid strand may also beincluded as described below.

The one or more polyphosphorolyzing agents may be included in thereaction mixture at any suitable concentration. For instance, a suitableconcentration may be approximately 1-500 μM. Other suitablepolyphosphorolyzing agent concentrations ranges may include but are notlimited to approximately 1-10 μM, 10-20 μM, 20-30 μM, 30-40 μM, 40-50μM, up to 50 μM, 50-60 μM, 60-70 μM, 70-80 μM, 90-100 μM, up to 100 μM,100-150 μM, 150-200 μM, up to 200 μM, 200-250 μM, 250-300 μM, up to 300μM, 300-350 μM, 350-400 μM, up to 400 μM, 400-450 μM, 450-500 μM.Additionally suitable polyphosphorolyzing agent concentrations includebut are not limited to 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85μM, 90 μM, 95 μM, 100 μM, 125 μM, 150 μM, 175 μM, 200 μM, 225 μM, 250μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, 400 μM, 425 μM, 450 μM, 475μM, and 500 μM. Particularly suitable concentrations ofpolyphosphorolyzing agent(s) may include but are not limited toapproximately 25 μM, 40 μM, 50 μM, and 100 μM, 150 μM, 200 μM and 250μM. Other suitable concentrations of polyphosphorolyzing agent may alsobe suitable as would be understood by one of skill in the art, and arealso contemplated to be part of this description.

The methods described herein may be carried out in any of severaldifferent forms. In some embodiments, the method comprises the followingsteps carried out serially:

-   -   (a) Annealing to the template strand a complementary activatable        oligonucleotide “P*”. This activatable oligonucleotide has a        non-extendable nucleotide at its 3′ terminus. It has no        nucleotides at or near its 3′ terminus that mismatch the        corresponding nucleotides on the template strand. Therefore, the        terminal nucleotide is hybridized to the template strand when        the oligonucleotide P* is annealed.    -   (b) Polyphosphorolyzing the annealed activatable oligonucleotide        P* with at least one polyphosphorolyzing agent described herein        and an enzyme that has polyphosphorolysis activity. This        activates the oligonucleotide P* by removal of the hybridized        terminal nucleotide.    -   (c) Polymerizing by extending the activated oligonucleotide P*        on the template strand in presence of four nucleoside        triphosphates and a nucleic acid polymerase to synthesize the        desired nucleic acid strand.        The APP method may also be used to amplify a desired nucleic        acid strand by, for example, adding the following additional        steps: (d) separating the desired nucleic acid strand of        step (c) from the template strand, and (e) repeating steps        (a)-(d) until a desired level of amplification of the desired        nucleic acid strand is achieved. Steps (a) to (c) of APP can be        conducted sequentially as two or more temperature stages on a        thermocycler, or they can be conducted as one temperature stage        on a thermocycler.

As described above, APP may be used to amplify nucleic acid molecules,including but not limited to ribonucleic acid (e.g., RNA) and/ordeoxyribonucleic acid (e.g., DNA). When used to amplify DNA, thenon-extendable, activatable oligonucleotide P* is typically a2′-deoxyoligonucleotide, the terminal deoxynucleotide may be a2′,3′-dideoxynucleotide, the four nucleoside triphosphates are2′-deoxynucleoside triphosphates, and the nucleic acid polymerase is aDNA polymerase. The DNA polymerase used in step (c) can also be theenzyme having polyphosphorolysis activity used in step (b).Amplification by APP may be linear or exponential. Linear amplificationis obtained when the activatable oligonucleotide P* is the onlycomplementary oligonucleotide used. Exponential amplification isobtained when a second oligonucleotide is present that is complementaryto the desired nucleic acid strand (e.g., as in PCR). The secondoligonucleotide can either be an extendable or an activatablenon-extendable oligonucleotide. The activatable oligonucleotide P* andthe second oligonucleotide flank the region that is targeted foramplification. In step (a), the second oligonucleotide anneals to theseparated desired nucleic acid strand product of step (d). In step (c),polymerization extends the second oligonucleotide on the desired nucleicacid strand to synthesize a copy of the nucleic acid template strand. Instep (d), the synthesized nucleic acid template strand is separated fromthe desired nucleic acid strand. Steps (a) through (d) may then berepeated until the desired level exponential amplification has beenachieved.

In certain embodiments, the APP method is used for allele-specificamplification. The nucleic acid template strand is typically a sense orantisense strand of one allele and is present in mixture with thecorresponding (sense or antisense) nucleic acid strand of the secondallele. The activatable (e.g., non-extendable) oligonucleotide P* has nomismatches near the 3′ terminus of the first allelic strand and has atleast one nucleotide at or near its 3′ terminus that mismatches thecorresponding nucleotide of the second allelic strand. Because of themismatch, in step (a) of the APP method the terminal non-extendablenucleotide of oligonucleotide P* is not hybridized to the second allele.In step (b), polyphosphorolysis does not substantially remove thenon-hybridized terminal or near terminal nucleotide from the activatableoligonucleotide P* annealed to the second allele. In step (c),therefore, the oligonucleotide P* is not substantially extended bypolymerization on the second allele. As a result, the desired nucleicacid strand of the first allele synthesized on the template strand isamplified preferentially over any nucleic acid strand synthesized on thesecond allele. In one embodiment, the APP method is used for exponentialamplification of a rare, mutant allele in a mixture containing one ormore wild-type alleles. Strands of the alleles may be separated toprovide single-stranded DNA, followed by the serial steps (a)-(e):

-   -   (a) Annealing to the sense or antisense strands of each allele a        complementary activatable 2′-deoxyoligonucleotide P* that has a        non-extendable 2′,3′-dideoxynucleotide at its 3′ terminus. P*        has no nucleotides at or near its 3′ terminus that mismatch the        corresponding 2′-deoxynucleotides on the mutant strand, but has        at least one nucleotide at or near its 3′ terminus that        mismatches the corresponding 2′-deoxynucleotide on the wild type        strand. Consequently, the terminal 2′,3′-dideoxynucleotide is        hybridized to the mutant strand but not to the wild-type strand        when the oligonucleotide P* is annealed. Simultaneously, a        second 2′-deoxyoligonucleotide that is complementary to the        anti-parallel strands of each allele is annealed to the        anti-parallel strands. The activatable 2′-deoxyoligonucleotide        P* and the second 2′-deoxyoligonucleotide flank the region of        the gene to be amplified.    -   (b) Polyphosphorolyzing the activatable P* that is annealed to a        mutant strand with at least one polyphosphorolyzing agent and an        enzyme that has polyphosphorolysis activity. This activates the        P* that is annealed to the mutant strand by removal of the        hybridized terminal 2′,3′-dideoxynucleotide. It does not        substantially activate the P* that is annealed to the wild-type        strand because the non-hybridized terminal        2′,3′-dideoxynucleotide is not substantially removed by the        polyphosporolysis.    -   (c) Polymerizing by extending the activated oligonucleotide P*        on the mutant strand in presence of four nucleoside        triphosphates and a DNA polymerase and simultaneously extending        the second 2′-deoxyoligonucleotide on both mutant and wild-type        anti-parallel strands.    -   (d) Separating the extension products of step (c);    -   (e) Repeating steps (a)-(d) until the desired level of        exponential amplification of the mutant allele has been        achieved.        The activatable P* is typically annealed to the antisense        strands of the alleles and the second P* is annealed to the        sense strands, or vice versa.

The methods described herein may also be used for scanning unknownsequence variants in a nucleic acid sequence or for re-sequencing of apredetermined sequence in a nucleic acid by carrying out the followingsteps serially:

-   -   (a) Mixing under hybridization conditions a template strand of        the nucleic acid with multiple sets of four activatable        oligonucleotides P* which are sufficiently complementary to the        template strand to hybridize therewith. Within each set, the        oligonucleotides P* differ from each other in having a different        3′-terminal non-extendable nucleotide, so that the 3′ terminal        non-extendable nucleotide is hybridized to the template strand        if the template strand is complementary to the 3′ terminal        non-extendable nucleotide. The number of sets corresponds to the        number of nucleotides in the sequence to be interrogated.    -   (b) Treating the resulting duplexes with at least one        polyphosphorolyzing agent described herein and an enzyme that        has polyphosphorolysis activity to activate by        polyphosphorolysis only those oligonucleotides P* which have a        3′ terminal non-extendable nucleotide that is hybridized to the        template strand.    -   (c) Polymerizing by extending the activated oligonucleotides P*        on the template strand in presence of four nucleoside        triphosphates and a nucleic acid polymerase.    -   (d) Separating the nucleic acid strands synthesized in step (c)        from the template strand.    -   (e) Repeating steps (a)-(d) until a desired level of        amplification is achieved, and    -   (f) Arranging the nucleic acid sequence in order by analyzing        overlaps of oligonuclotides P* that produced amplifications.

It is to be understood that the particular methods, reaction mixtures,and/or systems described herein may vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. Further, unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention pertains. As used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” also include pluralreferents unless the context clearly provides otherwise. All numericalranges are intended to encompass each individual value within the rangeas if each were separately listed (e.g., 10-20 may include one or moreof 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20). In terms ofconcentration ranges, these encompass fractional ranges, e.g., allvalues between 10-20 as if each were individually written out (e.g.,10.8, 11.5). The term “approximately”, when used to modify a group ofnumerical values, is meant to apply to each value individually unlessotherwise indicated.

An “amplicon” typically refers to a molecule made by copying ortranscribing another molecule, e.g., as occurs in transcription,cloning, and/or in a polymerase chain reaction (“PCR”) (e.g., stranddisplacement PCR amplification (SDA), duplex PCR amplification, etc.) orother nucleic acid amplification technique. Typically, an amplicon is acopy of a selected nucleic acid or a portion thereof (e.g., a templateor target nucleic acid) or is complementary thereto. The term“amplifying” or “amplification” in the context of nucleic acidstypically refers to the production of multiple copies of apolynucleotide, or a portion of the polynucleotide, typically startingfrom a small amount of the polynucleotide (e.g., a single polynucleotidemolecule), where the amplification products or amplicons are generallydetectable. Any of several methods may be used to amplify the targetnucleic acid from the sample. The term “amplifying” which typicallyrefers to an “exponential” increase in target nucleic acid is being usedherein to describe both linear and exponential increases in the numbersof a select target sequence of nucleic acid. The term “amplificationreaction mixture” refers to an aqueous solution comprising the variousreagents used to amplify a target nucleic acid. These include enzymes,aqueous buffers, salts, amplification primers, target nucleic acid, andnucleoside triphosphates. Depending upon the context, the mixture can beeither a complete or incomplete amplification reaction mixture. Themethod used to amplify the target nucleic acid may be any available toone of skill in the art. Any in vitro means for multiplying the copiesof a target sequence of nucleic acid may be utilized. These includelinear, logarithmic, and/or any other amplification method. Exemplarymethods include polymerase chain reaction (PCR; see, e.g., U.S. Pat.Nos. 4,683,202; 4,683,195; 4,965,188; and/or 5,035,996), isothermalprocedures (using one or more RNA polymerases (see, e.g., WO2006/081222), strand displacement (see, e.g., U.S. Pat. No. RE39007E),partial destruction of primer molecules (see, e.g., WO2006087574)),ligase chain reaction (LCR) (see, e.g., Wu, et al., Genomics 4: 560-569(1990) and/or Barany, et al. PNAS USA 88:189-193 (1991)), Qβ RNAreplicase systems (see, e.g., WO/1994/016108), RNA transcription-basedsystems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g.,U.S. Pat. No. 5,854,033; U.S. Pub. No. 2004/265897; Lizardi et al. Nat.Genet. 19: 225-232 (1998); and/or Banér et al. Nucleic Acid Res., 26:5073-5078 (1998)), and strand displacement amplification (SDA) (Little,et al. Clin Chem 45:777-784 (1999)), among others. Many systems aresuitable for use in amplifying target nucleic acids and are contemplatedherein as would be understood by one of skill in the art.

Any of several methods may be used to detect APP-amplified targetnucleic acids using various primers and/or probes. Many differentreagents, systems, and/or detectable labels may be used in the methodsdescribed herein. These include, for example, TaqMan® systems,detectable label-quencher systems (e.g., FRET, salicylate/DTPA ligandsystems (see, e.g., Oser et al. Angew. Chem. Int. Engl. 29(10):1167(1990), displacement hybridization, homologous probes, assays describedin EP 070685), molecular beacons (e.g., NASBA), Scorpion, locked nucleicacid (LNA) bases (Singh, et al. Chem Commum 4:455-456 (1998)), peptidenucleic acid (PNA) probes (Pellestor, et al. European J. Human Gen.12:694-700 (2004)), Eclipse probes (Afonina, et al. Biotechniques32:940-949 (2002)), light-up probes (Svanvik, et al. Anal Biochem281:26-35 (2001)), molecular beacons (Tyagi, et al. Nat. Biotechnol.14:303-308 (1996)), tripartite molecular beacons (Nutiu, et al. NucleicAcids Res. 30:e94 (2002)), QuantiProbes (www.qiagen.com), HyBeacons(French, et al. Mol. Cell. Probes 15:363-374 (2001)), displacementprobes (Li, et al. Nucleic Acids Res. 30:e5 (2002)), HybProbes(Cardullo, et al. PNAS 85:8790-8794 (1988)), MGB Alert(www.nanogen.com), Q-PNA (Fiandaca, et al. Genome Res. 11:609-611(2001)), Plexor (www.Promega.com), LUX primers (Nazarenko, et al.Nucleic Acids Res. 30:e37 (2002)), Scorpion primers (Whitcombe, et al.Nat Biotechnol 17:804-807 (1999)), AmpliFluor (Sunrise) primers(Nazarenko, et al. Nucleic Acids Res. 25:2516-2521 (1997)), DzyNAprimers (Todd, et al. Clin. Chem. 46:625-630 (2000)), and the like. Ineach of these assays, the generation of amplification products may bemonitored while the reaction is in progress. An apparatus for detectingthe signal generated by the detectable label may be used to detect,measure, and quantify the signal before, during, and/or afteramplification. The particular type of signal may dictate the choice ofdetection method. For example, in some embodiments, fluorescent dyes areused to label probes and/or amplified products. The probes bind tosingle-stranded and/or double-stranded amplified products, and/or thedyes intercalate into the double-stranded amplified products, andconsequently, the resulting fluorescence increases as the amount ofamplified product increases. In some embodiments, the T_(m) isascertained by observing a fluorescence decrease as the double-strandedamplified product dissociates and the intercalating dye is releasedtherefrom. The amount of fluorescence may be quantitated using standardequipment such as a spectra-fluorometer, for example. The use of othermethods and/or reagents is also contemplated herein as would beunderstood by one of skill in the art.

One exemplary method for amplifying and detecting target nucleic acidsis commercially available as TaqMan® (see, e.g., U.S. Pat. Nos.4,889,818; 5,079,352; 5,210,015; 5,436,134; 5,487,972; 5,658,751;5,210,015; 5,487,972; 5,538,848; 5,618,711; 5,677,152; 5,723,591;5,773,258; 5,789,224; 5,801,155; 5,804,375; 5,876,930; 5,994,056;6,030,787; 6,084,102; 6,127,155; 6,171,785; 6,214,979; 6,258,569;6,814,934; 6,821,727; 7,141,377; and/or 7,445,900). TaqMan® assays aretypically carried out by performing nucleic acid amplification on atarget polynucleotide using a nucleic acid polymerase having 5′-3′nuclease activity, a primer capable of hybridizing to said targetpolynucleotide, and an oligonucleotide probe capable of hybridizing tosaid target polynucleotide 3′ relative to said primer. Theoligonucleotide probe typically includes a detectable label (e.g., afluorescent reporter molecule) and a quencher molecule capable ofquenching the fluorescence of said reporter molecule. Typically, thedetectable label and quencher molecule are part of a single probe. Asamplification proceeds, the polymerase digests the probe to separate thedetectable label from the quencher molecule. The detectable label (e.g.,fluorescence) is monitored during the reaction, where detection of thelabel corresponds to the occurrence of nucleic acid amplification (e.g.,the higher the signal the greater the amount of amplification).Variations of TaqMan® assays (e.g., LNA™ spiked Taqman® assay) are knownin the art and would be suitable for use in the methods describedherein.

Another exemplary system utilizes double-stranded probes in displacementhybridization methods (see, e.g., Morrison et al. Anal. Biochem.,18:231-244 (1989); and/or Li, et al. Nucleic Acids Res., 30(2,e5)(2002)). In such methods, the probe typically includes two complementaryoligonucleotides of different lengths where one includes a detectablelabel and the other includes a quencher molecule. When not bound to atarget nucleic acid, the quencher suppresses the signal from thedetectable label. The probe becomes detectable upon displacementhybridization with a target nucleic acid. Multiple probes may be used,each containing different detectable labels, such that multiple targetnucleic acids may be queried in a single reaction.

Additional exemplary methods for amplifying and detecting target nucleicacids involve “molecular beacons”, which are single-stranded hairpinshaped oligonucleotide probes. In the presence of the target sequence,the probe unfolds, binds and emits a signal (e.g., fluoresces). Amolecular beacon typically includes at least four components: 1) the“loop”, an 18-30 nucleotide region which is complementary to the targetsequence; 2) two 5-7 nucleotide “stems” found on either end of the loopand being complementary to one another; 3) at the 5′ end, a detectablelabel; and 4) at the 3′ end, a quencher dye that prevents the detectablelabel from emitting a single when the probe is in the closed loop shape(e.g., not bound to a target nucleic acid). Thus, in the presence of acomplementary target, the “stem” portion of the beacon separates outresulting in the probe hybridizing to the target. Other types ofmolecular beacons are also known and may be suitable for use in themethods described herein. Molecular beacons may be used in a variety ofassay systems. One such system is nucleic acid sequence-basedamplification (NASBA®), a single step isothermal process for amplifyingRNA to double stranded DNA without temperature cycling. A NASBA reactiontypically requires avian myeloblastosis virus reverse transcriptase(AMV-RT or AMV), T7 RNA polymerase, RNase H, and two oligonucleotideprimers. After amplification, the amplified target nucleic acid may bedetected using a molecular beacon. Other uses for molecular beacons areknown in the art and would be suitable for use in the methods describedherein.

The Scorpion system is another exemplary assay format that may be usedin the methods described herein. Scorpion primers are bi-functionalmolecules in which a primer is covalently linked to the probe, alongwith a detectable label (e.g., a fluorophore) and a quencher. In thepresence of a target nucleic acid, the detectable label and the quencherseparate which leads to an increase in signal emitted from thedetectable label. Typically, a primer used in the amplification reactionincludes a probe element at the 5′ end along with a “PCR blocker”element (e.g., HEG monomer) at the start of the hairpin loop. The probetypically includes a self-complementary stem sequence with a detectablelabel at one end and a quencher at the other. In the initialamplification cycles (e.g., PCR), the primer hybridizes to the targetand extension occurs due to the action of polymerase. The Scorpionsystem may be used to examine and identify point mutations usingmultiple probes that may be differently tagged to distinguish betweenthe probes. Using PCR as an example, after one extension cycle iscomplete, the newly synthesized target region will be attached to thesame strand as the probe. Following the second cycle of denaturation andannealing, the probe and the target hybridize. The hairpin sequence thenhybridizes to a part of the newly produced PCR product. This results inthe separation of the detectable label from the quencher and causesemission of the signal. Other uses for molecular beacons are known inthe art and would be suitable for use in the methods described herein.

One or more detectable labels and/or quenching agents are typicallyattached to an oligonucleotide (e.g., P*), primer and/or probe. Thedetectable label may emit a signal when free or when bound to the targetnucleic acid. The detectable label may also emit a signal when inproximity to another detectable label. Detectable labels may also beused with quencher molecules such that the signal is only detectablewhen not in sufficiently close proximity to the quencher molecule. Forinstance, in some embodiments, the assay system may cause the detectablelabel to be liberated from the quenching molecule. Any of severaldetectable labels may be used to label the primers and probes used inthe methods described herein. As mentioned above, in some embodimentsthe detectable label may be attached to a probe, which may beincorporated into a primer, or may otherwise bind to amplified targetnucleic acid (e.g., a detectable nucleic acid binding agent such as anintercalating or non-intercalating dye). When using more than onedetectable label, each should differ in their spectral properties suchthat the labels may be distinguished from each other, or such thattogether the detectable labels emit a signal that is not emitted byeither detectable label alone. Exemplary detectable labels include, forinstance, a fluorescent dye or fluorophore (e.g., a chemical group thatcan be excited by light to emit fluorescence or phosphorescence),“acceptor dyes” capable of quenching a fluorescent signal from afluorescent donor dye, and the like. Suitable detectable labels mayinclude, for example, fluorosceins (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT(Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE;6-carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430,488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680,700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510,505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591,630/650-X, 650/665-X, 665/676, FL, FL ATP, FI-Ceramide, R6G SE, TMR,TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), coumarins (e.g.,7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPMmethylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA,methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g.,calcium crimson, calcium green, calcium orange, calcofluor white),Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18,5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescentproteins (e.g., green fluorescent protein (e.g., GFP. EGFP), bluefluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyanfluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescentprotein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs(e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/dabcyl, fluorescein/fluorescein, BODIPY FL/BODIPY FL,Fluorescein/QSY7 and QSY9), LysoTracker and LysoSensor (e.g.,LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker YellowHCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensorBlue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153,LysoSensor Yellow/Blue DND-160, LysoSensor Yellow/Blue 10,000 MWdextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g.,110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine(5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine RhodamineB, Phallicidine, Phalloidine, Red, Rhod-2, 5-ROX (carboxy-X-rhodamine),Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine(TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in,e.g., US Pub. No. 2009/0197254), among others as would be known to thoseof skill in the art. Other detectable labels may also be used (see,e.g., US Pub. No. 2009/0197254), as would be known to those of skill inthe art.

Nucleic acid binding agents may also be used to detect nucleic acidsamplified using the methods described herein. Many suitable detectablenucleic acid binding agents are available to one of skill in the art andmay be used alone or in combination with other agents and/or componentsof an assay system. Exemplary DNA binding agents may include, forexample, acridines (e.g., acridine orange, acriflavine), actinomycin D(Jain, et al. J. Mol. Biol. 68:21 (1972)), anthramycin, BOBO™-1,BOBO™-3, BO-PRO™-1, cbromomycin, DAPI (Kapuseinski, et al. Nuc. AcidsRes. 6(112): 3519 (1979)), daunomycin, distamycin (e.g., distamycin D),dyes described in U.S. Pat. No. 7,387,887, ellipticine, ethidium salts(e.g., ethidium bromide), fluorcoumanin, fluorescent intercalators asdescribed in U.S. Pat. No. 4,257,774, GelStar® (Cambrex Bio ScienceRockland Inc., Rockland, Me.), Hoechst 33258 (Searle and Embrey, 1990,Nuc. Acids Res. 18:3753-3762), Hoechst 33342, homidium, JO-PRO™-1, LIZdyes, LO-PRO™-1, mepacrine, mithramycin, NED dyes, netropsin,4′6-diamidino-α-phenylindole, proflavine, POPO™-1, POPO™-3, PO-PRO™-1,propidium iodide, ruthenium polypyridyls, S5, SYBR® Gold, SYBR® Green I(U.S. Pat. Nos. 5,436,134 and 5,658,751), SYBR® Green II, SYTOX blue,SYTOX green, SYTO® 43, SYTO® 44, SYTO® 45, SYTOX® Blue, TO-PRO®-1, SYTO®11, SYTO® 13, SYTO® 15, SYTO® 16, SYTO® 20, SYTO® 23, thiazole orange(Aldrich Chemical Co., Milwaukee, Wis.), TOTO™-3, YO-PRO®-1, and YOYO®-3(Molecular Probes, Inc., Eugene, Oreg.), among others. SYBR® Green I(see, e.g., U.S. Pat. Nos. 5,436,134; 5,658,751; and/or 6,569,927), forexample, has been used to monitor a PCR reaction by amplifying thetarget sequence in the presence of the dye, exciting the biologicalsample with light at a wavelength absorbed by the dye and detecting theemission therefrom; and, determining a melting profile of the amplifiedtarget sequence. The presence of amplified products and, therefore, thetarget sequence in the sample, may thereafter be determined by, forexample, performing a melting curve analysis (e.g., non-linear leastsquares regression of the sum of multiple gaussians). It is to beunderstood that the use of the SYBR® Green dye is presented as anexample, and that many such dyes may be used in the methods describedherein. Other nucleic acid binding agents may also be suitable as wouldbe understood by one of skill in the art.

Nucleic acids “hybridize” or “anneal” in a base-pairing interaction ofone polynucleotide with another polynucleotide (typically anantiparallel polynucleotide) that results in formation of a duplex orother higher-ordered structure, typically termed a hybridizationcomplex. The primary interaction between the antiparallelpolynucleotides is typically base specific, e.g., A/T and G/C, byWatson/Crick and/or Hoogsteen-type interactions. It is not a requirementthat two polynucleotides have 100% complementarity over their fulllength to achieve hybridization. In some aspects, a hybridizationcomplex can form from intermolecular interactions, or alternatively, canform from intramolecular interactions. Hybridization occurs due to avariety of well-characterized forces, including hydrogen bonding,solvent exclusion, and base stacking An extensive guide to nucleichybridization may be found in, Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, part I,chapter 2, “Overview of principles of hybridization and the strategy ofnucleic acid probe assays,” Elsevier (1993).

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction or assay. Toillustrate, an amplification reaction mixture generally includes asolution containing reagents necessary to carry out an amplificationreaction, and typically contains primers, a biocatalyst (e.g., a nucleicacid polymerase, a ligase, etc.), dNTPs, and a divalent metal cation ina suitable buffer. A reaction mixture is referred to as complete if itcontains all reagents necessary to carry out the reaction, andincomplete if it contains only a subset of the necessary reagents. Itwill be understood by one of skill in the art that reaction componentsare routinely stored as separate solutions, each containing a subset ofthe total components, for reasons of convenience, storage stability, orto allow for application-dependent adjustment of the componentconcentrations, and that reaction components are combined prior to thereaction to create a complete reaction mixture. Furthermore, it will beunderstood by one of skill in the art that reaction components arepackaged separately for commercialization and that useful commercialkits may contain any subset of the reaction or assay components, whichincludes the biomolecules of the invention.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is or can be divided (e.g., a functionalgroup, substituent group, or the like). For example, an oligonucleotidedescribed herein includes at least one donor moiety and/or at least oneacceptor moiety in certain embodiments.

The term “mutation” refers to a nucleic acid that has been altered inits nucleic acid sequence or an encoded protein product of a nucleicacid that has been altered in its amino acid sequence relative to anunaltered or native form of the nucleic acid or encoded protein product.Such alterations include, for example, point mutations or substitutions,deletions and insertions.

The term “nucleic acid” or “polynucleotide” refers to a polymer that canbe corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleicacid (DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as modified forms thereof,peptide nucleic acids (PNAs), locked nucleic acids (LNA™), and the like.In certain embodiments, a nucleic acid can be a polymer that includesmultiple monomer types, e.g., both RNA and DNA subunits. A nucleic acidcan be or can include, e.g., a chromosome or chromosomal segment, avector (e.g., an expression vector), an expression cassette, a naked DNAor RNA polymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, a primer, etc. A nucleic acid may be, e.g.,single-stranded, double-stranded, triple-stranded (and the like) and isnot limited to any particular length. Unless otherwise indicated, aparticular nucleic acid sequence optionally comprises or encodescomplementary sequences, in addition to any sequence explicitlyindicated.

Nucleic acids are not limited to molecules having naturally occurringpolynucleotide sequences or structures, naturally occurring backbones,and/or naturally occurring internucleotide linkages. For example,nucleic acids containing one or more carbocyclic sugars are alsoincluded within this definition (Jenkins et al. (1995) Chem. Soc. Rev.pp 169-176, which is incorporated by reference). To further illustrate,although a nucleic acid will generally contain phosphodiester bonds, insome cases nucleic acid analogs are included that have alternatebackbones. These may include, without limitation, phosphoramide(Beaucage et al. (1993) Tetrahedron 49(10): 1925 and the referencestherein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977)Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res. 14:3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J.Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) Chemica Scripta26:1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437and U.S. Pat. No. 5,644,048), phosphorodithioate (Brill et al. (1989) J.Am. Chem. Soc. 111:2321), O-methylphophoroamidite linkages (Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress (1992)), and peptide nucleic acid backbones and linkages (Egholm(1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed.Engl. 31:1008; Nielsen (1993) Nature 365:566; and Carlsson et al. (1996)Nature 380:207). Other analog nucleic acids include those withpositively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad.Sci. USA 92:6097); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghvi and P. Dan Cook,which are each incorporated by reference. Several nucleic acid analogsare also described in, e.g., Rawls, C & E News Jun. 2, 1997 page 35.Modifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties, such as labeling moieties, or toalter the stability and half-life of such molecules in physiologicalenvironments.

In addition to naturally occurring heterocyclic bases that are typicallyfound in nucleic acids (e.g., adenine, guanine, thymine, cytosine, anduracil), nucleic acid analogs also include those having non-naturallyoccurring heterocyclic or other modified bases. For instance, certainbases used in nucleotides that act as melting temperature (T_(m))modifiers are optionally included. Exemplary of these are 7-deazapurines(e.g., 7-deazaguanine, 7-deazaadenine, and the like);pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,and the like); hypoxanthine; inosine; xanthine; 8-aza derivatives of2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine,2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine,inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil; non-naturally occurring bases asdescribed by, for example, Seela et al. ((1991) Helv. Chim. Acta74:1790; (1999) Helv. Chim. Acta 82:1640); Grein et al. ((1994) Bioorg.Med. Chem. Lett. 4:971-976), U.S. Pat. Nos. 5,484,908, 5,645,985,5,990,303, 5,830,653, 6,639,059, 6,303,315, U.S. Pat. Appln. No.2003/0092905, and the like.

“Nucleoside” typically refers to a nucleic acid component that comprisesa base or basic group (e.g., comprising at least one homocyclic ring, atleast one heterocyclic ring, at least one aryl group, and/or the like)covalently linked to a sugar moiety (e.g., a ribose sugar, etc.), aderivative of a sugar moiety, or a functional equivalent of a sugarmoiety (e.g., an analog, such as carbocyclic ring). For example, when anucleoside includes a sugar moiety, the base is typically linked to a1′-position of that sugar moiety. As described above, a base can benaturally occurring (e.g., a purine base, such as adenine (A) or guanine(G), a pyrimidine base, such as thymine (T), cytosine (C), or uracil(U)), or non-naturally occurring (e.g., a 7-deazapurine base, apyrazolo[3,4-d]pyrimidine base, a propynyl-dN base, etc.). Exemplarynucleosides include ribonucleosides, deoxyribonucleosides,dideoxyribonucleosides, carbocyclic nucleosides, and the like. A“nucleotide” refers to an ester of a nucleoside, e.g., a phosphate esterof a nucleoside. To illustrate, a nucleotide can include 1, 2, 3, ormore phosphate groups covalently linked to a 5′ position of a sugarmoiety of the nucleoside. Nucleoside triphosphates and2′-deoxynucleoside triphosphates or their chemically modified versionsmay be used as substrates for multiple-nucleotide extension by APPwhere, for example, one nucleotide is incorporated the extending strandcan be further extended. 2′,3′-dideoxynucleoside triphosphates,chemically modified versions thereof, or other suitable compounds (e.g.,as in US 2005/0037398A1, acycloNMP) may be used as terminators forfurther extension may be used for single-nucleotide extension. Furtherexamples of 2′-terminated NTPs are described in U.S. Pat. No. 7,745,125B2, which is hereby incorporated by reference in its entirety.2′,3′-dideoxynucleoside triphosphates may be labeled with radioactivityor fluorescence dye for differentiation from the 3′ terminaldideoxynucleotide of oligonucleotide P*. Mixtures of nucleosidetriphosphates or 2′-deoxynucleoside triphosphates and2′,3′-dideoxynucleoside triphosphates may also be used.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein- and/or nucleic acid-based catalyst that acts to reduce theactivation energy of a chemical reaction involving other compounds or“substrates.” A “nucleotide incorporating enzyme” refers to an enzymethat catalyzes the incorporation of nucleotides into a nucleic acid,e.g., during nucleic acid amplification or the like. Exemplarynucleotide incorporating enzymes include, e.g., polymerases, terminaltransferases, reverse transcriptases (e.g., RQY polymerase describedabove), telomerases, polynucleotide phosphorylases, and the like. A“thermostable enzyme” refers to an enzyme that is stable to heat, isheat resistant, and retains sufficient catalytic activity when subjectedto elevated temperatures for selected periods of time. For example, athermostable polymerase retains sufficient activity to effect subsequentprimer extension reactions when subjected to elevated temperatures forthe time necessary to effect denaturation of double-stranded nucleicacids. Heating conditions necessary for nucleic acid denaturation arewell known to persons skilled in the art and are exemplified in, forexample, U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188. To furtherillustrate, a “thermostable polymerase” refers to an enzyme that issuitable for use in a temperature cycling reaction, such as a polymerasechain reaction (“PCR”). For a thermostable polymerase, enzymaticactivity refers to the catalysis of the combination of the nucleotidesin the proper manner to form primer extension products that arecomplementary to a template nucleic acid. Exemplary thermostablepolymerases are described herein, and others may available to theskilled artisan may also be suitable.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.Typically, the nucleoside monomers are linked by phosphodiester bonds oranalogs thereof, including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions arepresent. Typically, the 3′ end linkage of P* is a phosphodiester bond.Oligonucleotides may be prepared by any suitable method, including, butnot limited to, isolation of an existing or natural sequence, DNAreplication or amplification, reverse transcription, cloning andrestriction digestion of appropriate sequences, or direct chemicalsynthesis by a method such as the phosphotriester method of Narang etal. ((1979) Meth. Enzymol. 68:90-99); the phosphodiester method of Brownet al. ((1979) Meth. Enzymol. 68:109-151); the diethylphosphoramiditemethod of Beaucage et al. ((1981) Tetrahedron Lett. 22:1859-1862); thetriester method of Matteucci et al. ((1981) J. Am. Chem. Soc.103:3185-3191); automated synthesis methods; or the solid support methoddescribed in U.S. Pat. No. 4,458,066, and/or other methods known tothose skilled in the art.

As described above, “P*” represents an oligonucleotide having anon-extendable 3′ end (“P*”) that is removable by polyphosphorolysis. AP* oligonucleotide may also be referred to as “activatable”, in thatremoval of the non-extendable 3′end renders the oligonucleotide suitablefor polymerization (e.g., “activated P*”). The non-extendable 3′ end ofa P* oligonucleotide may be a dideoxynucleotide, an acyclonuclotide, aterminator having a 3′-hydroxyl but being 2′ modified with a bulkymolecule to prevent extension from the 3′-OH (e.g., US 2007/0154914A1),or a “virtual” terminator having modification(s) at the base to preventextension from the 3′OH′ by AmpliTaqFS (Wu, et al Nucleic AcidsResearch, 35(19): 6339-6349 (2007)). Other forms of P* are alsocontemplated and may be of use in the methods described herein, as wouldbe understood by one of skill in the art.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a target or template nucleic acid and permit chain extension orelongation using, e.g., a nucleotide incorporating biocatalyst, such asa polymerase under appropriate reaction conditions. A primer nucleicacid is typically a natural or synthetic oligonucleotide (e.g., asingle-stranded oligodeoxyribonucleotide). Although other primer nucleicacid lengths are optionally utilized, they typically comprisehybridizing regions that range from about 8 to about 100 nucleotides inlength. Shorter primer nucleic acids generally require lowertemperatures to form sufficiently stable hybrid complexes with templatenucleic acids. A primer nucleic acid that is at least partiallycomplementary to a subsequence of a template nucleic acid is typicallysufficient to hybridize with the template for extension to occur. Aprimer nucleic acid may be labeled, if desired, by incorporating adetectable label as described herein.

The term “probe nucleic acid” or “probe” refers to a labeled orunlabeled oligonucleotide capable of selectively hybridizing to a targetor template nucleic acid under suitable conditions. Typically, a probeis sufficiently complementary to a specific target sequence contained ina nucleic acid sample to form a stable hybridization duplex with thetarget sequence under selected hybridization conditions. A hybridizationassay carried out using a probe under sufficiently stringenthybridization conditions permits the selective detection of a specifictarget sequence. The term “hybridizing region” refers to that region ofa nucleic acid that is exactly or substantially complementary to, andtherefore capable of hybridizing to, the target sequence. For use in ahybridization assay for the discrimination of single nucleotidedifferences in sequence, the hybridizing region is typically from about8 to about 100 nucleotides in length. Although the hybridizing regiongenerally refers to the entire oligonucleotide, the probe may includeadditional nucleotide sequences that function, for example, as linkerbinding sites to provide a site for attaching the probe sequence to asolid support. A probe of the invention may be generally included in anucleic acid that comprises one or more labels (e.g., donor moieties,acceptor moieties, and/or quencher moieties), such as a 5′-nucleaseprobe, a hybridization probe, a fluorescent resonance energy transfer(FRET) probe, a hairpin probe, or a molecular beacon, which can also beutilized to detect hybridization between the probe and target nucleicacids in a sample. In some embodiments, the hybridizing region of theprobe is completely complementary to the target sequence. However, ingeneral, complete complementarity is not necessary (e.g., nucleic acidscan be partially complementary to one another); stable hybridizationcomplexes may contain mismatched bases or unmatched bases. Modificationof the stringent conditions may be necessary to permit a stablehybridization complex with one or more base pair mismatches or unmatchedbases. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)provides guidance for suitable modification. Stability of thetarget/probe hybridization complex depends on a number of variablesincluding length of the oligonucleotide, base composition and sequenceof the oligonucleotide, temperature, and ionic conditions. One of skillin the art will recognize that, in general, the exact complement of agiven probe is similarly useful as a probe. One of skill in the art willalso recognize that, in certain embodiments, probe nucleic acids canalso be used as primer nucleic acids.

The methods described herein may be useful for detecting and/orquantifying a variety of target nucleic acids from a test sample. Atarget nucleic acid is any nucleic acid for which an assay system isdesigned to identify or detect as present (or not), and/or quantify in atest sample. Such nucleic acids may include, for example, those ofinfectious agents (e.g., virus, bacteria, parasite, and the like), adisease process such as cancer, diabetes, or the like, or to measure animmune response. Exemplary “test samples” include various types ofsamples, such as biological samples. Exemplary biological samplesinclude, for instance, a bodily fluid (e.g., blood, saliva, or spinalfluid), a tissue sample, a food (e.g., meat) or beverage (e.g., milk)product, or the like. Expressed nucleic acids may include, for example,genes for which expression (or lack thereof) is associated with medicalconditions such as infectious disease (e.g., bacterial, viral, fungal,protozoal infections) or cancer. The methods described herein may alsobe used to detect contaminants (e.g., bacteria, virus, fungus, and/orprotozoan) in pharmaceutical, food, or beverage products. The methodsdescribed herein may be also be used to detect rare alleles in thepresence of wild type alleles (e.g., one mutant allele in the presenceof 10⁶-10⁹ wild type alleles). The methods are useful to, for example,detect minimal residual disease (e.g., rare remaining cancer cellsduring remission, especially mutations in the p53 gene or other tumorsuppressor genes previously identified within the tumors), and/ormeasure mutation load (e.g., the frequency of specific somatic mutationspresent in normal tissues, such as blood or urine).

Kits for performing the methods described herein are also provided. Thekit typically includes at least a pair of oligonucleotides (e.g., atleast one of the pair being a P* oligonucleotide) for amplifying atleast one target nucleic acid from a sample, one or morepolyphosphorolyzing agents described herein, a biocatalyst (e.g., DNApolymerase) and/or corresponding one or more probes labeled with adetectable label. The kit may also include samples containingpre-defined target nucleic acids to be used in control reactions. Thekit may also optionally include stock solutions, buffers, enzymes,detectable labels or reagents required for detection, tubes, membranes,and the like that may be used to complete the amplification reaction. Insome embodiments, multiple primer sets are included. Other embodimentsof particular systems and kits are also contemplated which would beunderstood by one of skill in the art.

Certain embodiments are further described in the following examples.These embodiments are provided as examples only and are not intended tolimit the scope of the claims in any way. All references cited withinthis application are incorporated by reference in their entirety intothis application.

EXAMPLES Example 1 Titration of Human DNA with Triphosphate

Human DNA template titrations were performed with and without thepresence of human genomic DNA. FIG. 1 shows the amplification curves inthe presence of 250 ng, 25 ng, 2.5 ng, 250 pg, and 25 pg of human DNAand non-template control (NTC) (to test for non-specific amplification).These data illustrate that dye-based detection of amplification may beachieved using triphosphate-catalyzed reactions. The reactions wereperformed using an ABI 7900 Sequence Detection System with the followingtemperature profile: 50 cycles of 95° C. for 3 seconds, then 65° C. for60 seconds. Each 20 μl-reaction contained 50 mM Tris (pH 8.25), 1×EvaGreen, 31 μM dNTPs, 3 mM MgCl₂, 60 mM KCl, Tween 0.1%, 40 μMtriphosphate (pentasodium triphosphate, Sigma-Aldrich cat. No. 72061),10 pg/μl Taq FY mutant DNA polymerase 10A per 100 μl reaction, 500 nMPA-1F (5′-CATCCTGGTTTGTGTTTTGCCTAA(ddC)-3′; SEQ ID NO.: 1), 500 nM PA-1R(5′-GGGAGAAAAAAGCCAACCTTAATG(ddC)-3′ SEQ ID NO.: 2), and Human DNA (Cat.No. #403062, Life Technologies, Carlsbad, Calif.) at the designatedconcentrations.

Example 2 Titration of Human DNA Using Power SYBR

Human DNA template titrations were performed with and without thepresence of human genomic DNA with ABI's Power SYBR. FIG. 2 shows theamplification curves using Power SYBR (dash lines) in the presence of250 ng, 25 ng, 2.5 ng, 250 pg, and 25 pg of human DNA and non-templatecontrol (NTC) (to test for non-specific amplification) in comparisonwith triphosphate-catalyzed reactions (solid lines) performed asdescribed in Example 1. These data illustrate that thetriphosphate-catalyzed reactions may provide improved C_(T) and/orsignal strength. The SYBR Green reactions were performed using an ABI7900 Sequence Detection System with the following temperature profile:50 cycles of 95° C. for 3 seconds, then 60° C. for 60 seconds. Each 20μL-reaction contained 1× Power SYBR master mix (Cat. No. #4385372, LifeTechnologies, Carlsbad, Calif.), 500 nM PA-1F(5′-CATCCTGGTTTGTGTTTTGCCTAAC-3′; SEQ ID NO.: 1), 500 nM PA-1R(5′-GGGAGAAAAAAGCCAACCTTAATGC-3′ SEQ ID NO.: 2), and Human DNA (Cat. No.#403062, Life Technologies, Carlsbad, Calif.) at the designatedconcentrations.

Example 3 Amplification of a Plasmid Template by Power SYBR

A titration of a plasmid DNA was performed with ABI's Fast SYBR. Theresult showed that the master mix did not yield a specific product.FIGS. 3A-C shows the amplification curves using Fast SYBR in thepresence of 1 μL of undiluted plasmid prep (trace 1), 10-fold dilutedplasmid prep (trace 2), 100 fold diluted plasmid prep (trace 3), 1000fold diluted plasmid prep (trace 4), and a NTC (trace 5) performed asdescribed following a thermal profile as described in Example 1. Meltingcurves are shown in FIG. 3B, in which traces 6, 7, 8, 9, and 10correspond to the amplifications represented by traces 1-5,respectively, of FIG. 3A. FIG. 3C illustrates an agarose gelelectrophoresis image of the PCR reactions shown by traces 1 (Lane 7C1)and 5 (Lane 7C2). The Fast SYBR reactions were performed using an ABI7900 Sequence Detection System with the following temperature profile:40 cycles of 95° C. for 3 seconds, then 60° C. for 60 seconds. Each 20μl reaction contained 1× Fast SYBR master mix, 500 nM vD-primer:TGCAATACCGTGAGCTGACCC (SEQ ID NO.: 3), 500 nM v1699LR primer:GTCTGGTTGAAACGAGTGTGCAGGC (SEQ ID NO.: 4), and about ten million copiesof p540D plasmid DNA (a pUC-based plasmid containing an insert of thesequence of SEQ ID NO.: 7). The sequence amplified from plasmid 540D isTGCAATACCGTGAGCTGACCCgtctgcgttcgacctacatcgacccactgccggacctgattcatccacgtaccggccGCCTGCACACTCGTTTCAACCAGAC (SEQ ID NO.: 5) (primers bind atcapitalized sequence).

Example 4 Triphosphate-Catalyzed APP Amplification from Plasmid DNA

In contrast to the SYBR Green I reaction described in Example 3,triphosphate-catalyzed amplification of the aforementioned plasmid DNA(p540D) using ddC-terminated primers yields specific product. Morespecifically, the ddC-terminated primers for the reactions areTGCAATACCGTGAGCTGACC(ddC) (D primer, SEQ ID NO.: 3) and

GTCTGGTTGAAACGAGTGTGCAGG(ddC) (1699LR primer, SEQ ID NO.: 4). Each 20μL-reaction contained 50 mM Tris (pH 8), 1× EvaGreen, 31 μM dNTP, 2.5 mMMgCl₂, Tween 0.1%, 50 μM triphosphate, 10 pg/μl 10A (a F667Y mutation ofTaq DNA polymerase) per 100 μl reaction, 500 nM D-primer, 500 nM 1699LRprimer, and about ten million copies of p540D plasmid DNA. Thetriphosphate-catalyzed reactions were performed using an ABI 7900Sequence Detection System with the following temperature profile: 40cycles of 95° C., 3 seconds, then 65° C., 60 seconds. The results areshown in FIGS. 4A-C. The amplification curve from plasmid DNA is shownby Trace 11 in FIG. 4A, and the melting curve is shown by Trace 13 inFIG. 4B. The specific product generated by the reaction is shown in Lane8C1 of an agarose gel electrophoresis in FIG. 4C. The resultsdemonstrated that triphosphate-catalyzed reaction is more specific andstringent than the PowerSYBR PCR Products (e.g., as shown in FIGS.3A-C).

Example 5 Comparison of Triphosphate-Catalyzed Reactions andPPi-Catalyzed Reactions

A comparison between triphosphate and PPi was performed using titrationsof human DNA using primers PA-10F (GCATAGCAGTCCCCAAGAATGA (ddC); SEQ IDNO.: 6) and PA10R (CGGTTCCCACGAAAAGCAAC (ddC); SEQ ID NO.: 7). Theseprimers were designed to generate a 127 bp amplicon from the human DNAtemplate. Each 20 μl triphosphate-catalyzed reaction contained 50 mMTris (pH 8); 1× EvaGreen; 31 uM dNTP; 2.5 mM MgCl₂; Tween 0.1%; 25, 50,100, or 150 uM triphosphate; 10 pg/μl 10A per 100 uL reaction; 500 nMPA-10F; 500 nM PA-10R; and 125 ng, 12.5 ng, 1.25 ng, 125 pg, or 0 ng ofhuman DNA. PP_(i)-catalyzed reactions were identically designed as thetriphosphate-reactions except that PP_(i) was used instead oftriphosphate. The titration reactions were performed using an ABI 7900Sequence Detection System with the following temperature profile: 40cycles of 95° C., 3 seconds, then 60° C., 60 seconds. A melt analysiswas performed following amplification by PCR. The C_(T) of the titrationusing the various concentrations of PP_(i) and triphosphate are plottedin FIG. 5. PP_(i) showed inhibition at concentrations of 100 μM and 150μM, while triphosphate did not show significant inhibition at any ofthese tested concentrations. The amplification curves under 50 μMtriphosphate and 50 μM of PP_(i) are shown in FIGS. 6A/C and 6B/D/E,respectively. The melting curves of the triphosphate reactions (FIG. 6C)demonstrate that all products were clean products. In contrast, themelting curves of PP_(i) reaction for 1.25 ng human DNA (FIG. 6E) and125 pg human DNA (FIG. 6F) exhibit non-specific peaks in addition to thespecific peak. It should be noted that 50 μM PPi gave the most efficientreactions among the concentrations of PP_(i) tested. However, thisconcentration gave non-specific reactions at low input DNA. At 25 μMPPi, non-specific product was observed for 125 pg input human DNA (FIG.7). On the other hand, 100 and 150 μM PP_(i) greatly inhibitamplifications when the input DNA concentrations were low. For thisamplicon, PP_(i) did not exhibit an optimal concentration at which thereaction was both efficient and specific.

Example 6 Comparison of Triphosphate-Catalyzed Reactions andPPi-Catalyzed Reactions

A comparison between triphosphate and PP_(i) was performed usingtitrations of human DNA using primers PA-17F(GCTCCAGACAGAAACACCGTA(ddC); SEQ ID NO.: 8) and PA-17R(CCATAACCAGACTCAGCAGAGAA(ddC); SEQ ID NO.: 9). The primers were designedto generate a 108 bp amplicon from the human DNA template.Triphosphate-reactions and PPi-reactions were conducted as described forthe reactions of Example 5. The C_(T) of the titration at the variousconcentrations of PP_(i) and triphosphate are shown in FIG. 8. PP_(i)exhibited inhibition at concentrations of 100 μM and 150 μM asdemonstrated by the failed reactions in low input DNA, whiletriphosphate did not show significant inhibition at these testedconcentrations.

Example 7 Comparison of Triphosphate-Catalyzed and PPi-CatalyzedReactions

Linear amplifications are a convenient way to compare reaction rates.Here we employed fluorogenic substrates which generate fluorogenicsignals once they were converted from partial duplex to substantiallyfull duplex. This example demonstrated that triphosphate-initiateddeblocking is more stringent than PP_(i)-initiated deblocking reactions.

Four substrates described in this example were made as detailed below:

-   -   (1) Primer vPA1F (5′-CATCCTGGTTTGTGTTTTGCCTAAC-3′; SEQ ID        NO.: 1) was allowed to anneal to PAPbttm        (5′-/TAM/CAGCGTGTGGTATGCG/FAM-dT/CGACGTTAGGCAAAACACAAACCAGGATG-3′;        SEQ ID NO.: 10) to form a duplex as shown below as vSub(PM):

-   -   (2) Similarly, Primer vPA1(ms1)F        (5′-CATCCTGGTTTGTGTTTTGCCTAAT-3′; SEQ ID NO.: 11) was allowed to        anneal to PAPbttm to form a duplex as shown below as        vSub(ms@-1):

-   -   (3) Similarly, Primer PA1F (5′-CATCCTGGTTTGTGTTTTGCCTAA/ddC/-3′;        SEQ ID NO.: 1) was allowed to anneal to PAPbttm to form a duplex        as shown below as Sub(PM):

-   -   (4) Similarly, Primer PA1F was allowed to anneal to PAPbttm-T        (5′-/TAM/CAGCGTGTGGTATGCG/FAM-dT/CGACTTTAGGCAAAACACAAACCAGGATG-3′;        SEQ ID NO.: 12) to form a duplex as shown below as Sub(ms@-1):

The primers in vSub(PM) and vSub(ms@-1) are not blocked. The twosubstrates were used for regular reactions. The primers in Sub(PM) andSub(ms@-1) are blocked. The two substrates were used for linearreactions that involve deblocking and extension referred as coupledreactions.

Each linear amplification assay contained 50 mM Tris, pH 8 (Teknova,Hollister, Ca), 2.5 mM MgCl₂ (ABI, Foster City, Calif.), 0.1% Tween 20(Thermo Fisher, Rockford, Ill.), 0.25 mM total dNTP (ABI, Foster City,Calif.), 500 nM substrate, about 1 uM of ROX (Molecular Probes, Eugene,Oreg.), 150 μM tripolyphosphate (Cat #T5633, Sigma-Aldrich, St, Louis,Mo.) for triphosphate reactions or 30 μM pyrophosphate forPPi-reactions, and none of triphosphate or PPi for vanilla reactions,and 16.76 pg/μL 10A for vanilla reactions or 1.47 ng/μL 10A fortriphosphate- or PPi-initiated reactions. The reaction time courses oftriphosphate-initiated reactions with Sub(PM) and Sub(ms@-1) are shownin FIG. 9A.

In FIG. 9B, the reaction rates relative to the rate obtained with theirrespective perfectly matched substrate are tabulated. For the vanillareaction, the rate obtained using vSub(ms@-1) was 28% of the rateobtained using vSub(PM). In contrast, the rate obtained using Sub(ms@-1)was only 0.98% of the rate obtained using Sub(PM) for the coupledreaction where PPi was used to deblock the primer. The vast differencein reaction rate could explain the stringent reaction reported earlier.

Furthermore, the rate obtained using Sub(ms@-1) was 0.46% of the rateobtained using Sub(PM) when triphosphate was used to deblock thereaction. While the PPi reaction exhibited an error rate of 98 per10,000 correct starts, triphosphate-catalyzed reactions exhibited anerror rate of only 46 per 10,000 correct starts. Thus,triphosphate-initiated deblocking reactions unexpectedly exhibitimproved stringency over PPi-initiated deblocking reactions, consistentwith PCR results shown in Example 5 and Example 6. Therefore,triphosphate-catalyzed reactions are not due to the conversion oftriphosphate to PPi.

Example 8 Deblocking and Extension with Triphosphate

Therminator III DNA polymerase is a 9° N_(m) DNA polymerase variant withan enhanced ability to incorporate modified substrates such asdideoxynucleotides (Gardner, A F and Jack, W E, 1999, NAR 27:2545-2555;Gardner, A F and Jack, W E, 2002, NAR 30: 605-613). It belongs to thePol B family. This example demonstrated that triphosphate-initiateddeblocking was effective using Therminator III.

In the experiment, Primer PA16 (5′-GATCCTTTAGTGCTGGACTGACC-3′; SEQ IDNO.: 67) was first allowed to anneal to PA16Fbottm(5′-/TAM/CAGCGTGTGGTATGCG/FAM-dT/CGACGGTCAGTCCAGCACTAAAGGATC-3′; SEQ IDNO.: 13) to form a duplex as shown below as Sub(PA16F):

Then, the substrate at final concentration of 500 nM was allowed toreact at 65° C. with Therminator III (final concentration at 0.027unit/4) in 1× ThermoPol Buffer (provided by New England BioLabs) withadded dNTP to final 0.25 mM and 0, 50, 150, or 250 μM of triphosphate.The reaction time courses are shown in FIG. 10. The fluorescent changerate accelerated as the concentration of triphosphate increased.

Example 9 Deblocking and Extension by RQY in the Presence ofTriphosphate and Mn²⁺

RQ1 is a thermostable DNA polymerase from Thermus thermophilus (U.S.Pat. No. 7,422,872, incorporated herein by reference in its entirety).It belongs to Pol A family. The Sub(PM) in Example 7 at finalconcentration of 500 nM was used in reactions that contained 50 mMTris/HCl, pH8.0, 2.5 mM MnOAC₂, triphosphate and either 250 or 100 μMdNTPs and the RQY polymerase (a mutant of RQ1 including the substitutionof phenylalanine 669 with tyrosine). The reaction time courses are shownin FIG. 11. It is clear that RQY is able to deblock ddC in the presenceof triphosphate and Mn²⁺, and extend the activated primer to generatefluorescence change. Without triphosphate, the deblocking was noteffected.

Example 10 Tetraphosphate is an Effective Deblocking Reagent

Tetraphosphate is a polyphosphate that follows the formula:

where n=2 (FIG. 12B). This example is to demonstrate that tetraphosphateat 33 μM is an effective deblocking agent and is useful in APP.

The 20 μl-reaction contained 50 mM Tris/HCl, pH8.5, 2. mM MgCl₂, 33 μMtetraphosphate, 1.5×SYBR Green, 31 μM dNTP, 15 mM (NH₃)₂SO₄, Tween 0.1%,2 pg/μl 10A per 20 μl reaction, 500 nM PA-1F(5′-CATCCTGGTTTGTGTTTTGCCTAA(ddC)-3′; SEQ ID NO.: 1), 500 nM PA-1R(5′-GGGAGAAAAAAGCCAACCTTAATG(ddC)-3′ SEQ ID NO.: 2), and a total of 20ng of human DNA. The negative control reaction contained notetraphosphate. The tetraphosphate-catalyzed reactions were performedusing an ABI 7900 Sequence Detection System with the followingtemperature profile: 50 cycles of 95° C., 3 seconds, then 65° C., 60seconds. The result is shown in FIG. 12A. The reaction withtetraphosphate exhibited a positive amplification curve, while withouttetraphosphate, the reaction did not take off in fifty cycles.

Example 11 APP Using Imidodiphosphate (IDP)

IDP (imidodiphosphate, Sigma-Aldrich Cat #, CAS #26039-10-1; FIG. 13B)has a molecular formula showing below:

This example demonstrates that IDP is useful in APP.

The 20 μl-reaction contained 50 mM Tris/HCl, pH8.25, 1.5 mM MgCl₂, 62.5μM dNTP, 0.1% Tween 20, 5 unit of TaqFS, 500 nM SBTaqGn1598Fmu, 500 nM1699LR (SEQ ID NO.: 9), about 1 million copies of p540D with insert thathas SEQ ID NO.: 7 and with (a) final 1/4860 dilution of saturated IDPsolution, or (b) 1/18580 dilution of saturated IDP solution or (c) noIDP.

SBTaqGn1598Fmu has the sequence as5′-(BHQ)-TGCAATACCGTGAGC(FAM-dT)GACC(ddC) (SEQ ID NO.:14). TheIDP-catalyzed reactions were performed using an ABI 7900 SequenceDetection System with the following temperature profile: 50 cycles of95° C., 3 seconds, then 65° C., 120 seconds. The PCR products were runon an Ethidium Bromide Agarose gel (1%) as shown in FIG. 13A. Onlyreactions with IDP generated the specific product while reaction with noIDP generated no products.

Example 12 Pentaphosphate and Hexaphosphate are Effective DeblockingReagents

Pentaphosphate is a polyphosphate that follows the formula:

This example is to demonstrate that pentaphosphate is an effectivedeblocking agent and is useful in carrying out an APP reaction.

The 20 μl-reaction contains 50 mM Tris/HCl, pH8.5, 2. mM MgCl₂, 33 μMpenta- or hexa-phosphate, 1.5×SYBR Green, 31 μM dNTP, 15 mM (NH₃)₂SO₄,Tween 0.1%, 2 pg/μl 10A per 20 μl reaction, 500 nM PA-1F(5′-CATCCTGGTTTGTGTTTTGCCTAA(ddC)-3′; SEQ ID NO.: 1), 500 nM PA-1R(5′-GGGAGAAAAAAGCCAACCTTAATG(ddC)-3′ SEQ ID NO.: 2), and a total of 20ng of human DNA. The negative control reaction contains nopentaphosphate. The pentaphosphate-catalyzed reactions are performedusing an ABI 7900 Sequence Detection System with the followingtemperature profile: 50 cycles of 95° C., 3 seconds, then 65° C., 60seconds. The reaction is expected to show positive amplification curve.

Example 13 Preventing Primer-Dimer Formation

PCR is prone to form primer dimers when 3′ regions of the primers arecomplementary to or partially-complementary to each other. This exampleserves to demonstrate that APP is an effective tool to reduce primerdimer formation in PCR even when substantial complementarities existbetween primers.

Table I lists primers and their sequences used in this example toevaluate the potentials to form primer dimers. Regular primers vPA17PD4F(SEQ ID NO.: 16), vPA17PD6F (SEQ ID NO.: 17), and vPA17PD9F (SEQ ID NO.:18) are synthesized so that they make 4, 6, and 9 base pairs with vPA17R(SEQ ID NO.: 15) at 3′ ends. PA17PD4F, PA17PD6F, and PA17PD9F, and PA17Rare the APP counterparts with dideoxy endings. The underlines in Table Iindicate the complementary region to the corresponding reverse primers.

TABLE I Primer Sequences and Ct's in Primer-dimer Formation AssaysPrimer Primer Sequence SEQ ID Number Ct in PCR* vPA17RCCATAACCAGACTCAGCAGAGAAC SEQ ID NO.: 15 — vPA17PD4FGCTCCAGACAGAAACACCGTTC SEQ ID NO.: 16 33 vPA17PD6FGCTCCAGACAGAAACAGTTCTC SEQ ID NO.: 17 19 vPA17PD9FGCTCCAGACAGAAGTTCTCTGC SEQ ID NO.: 18 2 PA17RCCATAACCAGACTCAGCAGAGAA/ddC/ SEQ ID NO.: 15 — PA17PD4FGCTCCAGACAGAAACACCGTT/ddC/ SEQ ID NO.: 16 40 PA17PD6FGCTCCAGACAGAAACAGTTCT/ddC/ SEQ ID NO.: 17 34 PA17PD9FGCTCCAGACAGAAGTTCTCTG/ddC/ SEQ ID NO.: 18 29 *— Ct in PCR when pairedwith the corresponding forward primer

The regular PCR reaction of 20 μL contains 1× Fast SYBR Green Master Mix(Life Technologies, ABI Cat #4385612), and 500 nM of forward and reverseprimers, with each primer pair having 4, 6 and 9 bases overlapsrespectively. Assays were performed using an ABI 7900 Sequence DetectionSystem with the following temperature profile: 40 cycles of 95° C., 3seconds, then 65° C., 60 seconds. The reaction profiles are shown inFIG. 14. Traces 30, 31, and 32 are the regular reactions of 4, 6, and 9base-pair overlaps.

The APP reactions of 20 μL contains 50 mM Tris/HCl, pH8.5, 2.5 mM MgCl₂,350 μM triphosphate, 0.5×SYBR Green, 31 μM dNTP, 15 mM (NH₃)₂SO₄, Tween0.1%, 6 ng/μl 10A, 500 nM each of forward and reverse primers of 4, 6and 9 bases overlaps respectively. Assays were performed using an ABI7900 Sequence Detection System with the following temperature profile:40 cycles of 95° C., 3 seconds, then 65° C., 60 seconds. The reactionprofiles are shown in FIG. 14. Traces 33, 34, and 35 are APP reactionsby APP primers of 4, 6, and 9 base-pairs overlaps.

The Ct's of regular reaction and APP reaction are 33 and 40 respectivelyfor 4-base overlapping reactions, showing a 128 fold decrease inpotential of primer dimer formation in the APP reaction (assuming 2 folddecrease for every 1 cycle delay). The Ct's of regular reaction and APPreaction are 19 and 34 respectively for 6-base overlapping reactions,showing a near 33 thousands fold decrease in potential of primer dimerformation in the APP reaction. The Ct's of regular reaction and APPreaction are 2 and 29 respectively for 9-base overlapping reactions,showing an over 130 million fold decrease in potential of primer dimerformation in the APP reaction.

Example 14 Primer-Dimer Formation in Multiplex Assays

In multiplex reactions, the chance to form primer dimer increases as thenumber of primers increases. A regular multiplex PCR reaction of 20 μL,cycling between 95° C. for 5 second and 65° C. for 1 minute, contained1× Power SYBR Green Master Mix (Life Technologies, ABI Cat #4368706) and104 nM each of the primers listed in Table II, nonspecific signals cameout at cycle of 31 (Trace 36, FIG. 15). When the primer sequences werekept the same but the last bases were changed to terminators (TableIII), and the amplification was carried in APP multiplex reaction (20 uLof 50 mM Tris/HCl, pH8.5, 2.5 mM MgCl₂, 300 μM triphosphate, 0.5×SYBRGreen, 31 μM dNTP, 15 mM (NH₃)₂SO₄, Tween 0.1%, 6 ng/μl 10A), thenon-specific reaction did not show up in 40 cycles (Trace 37, FIG. 15).

TABLE II Primer Pools for regular and APP reactions Primer SequencesSEQ ID NO. CATCCTGGTTTGTGTTTTGCCTAAC 19 GACAATACTGTTCCAATTTGGCTCATC 20GCTCCAGTTGTATCATCCCCTTTTC 21 CACAAAGACACGAAGAACATCATTGAC 22GGGATGGCTCTATGGACAAAAAGAC 23 TGCCATGGTGCTTCATACAAGTATC 24CCCGGTTAAATACCATGAGATTCTAAGC 25 GGAGCAAGCTTGAAGGAGTTAGAATC 26CCATCAGGGAAGACTATCCTCAAAC 27 GCATAGCAGTCCCCAAGAATGAC 28CTCGCCATCCCCACCATAC 29 CCTCTTCATCCTCACCCTTGC 30 CCTGCCCTTCAGAACCTAACAC31 TCAGCACACCTATCGGAAAGC 32 GCGGATGCCTCCTTTGC 33 GATCCTTTAGTGCTGGACTGACC34 GCTCCAGACAGAAACACCGTAC 35 ATCCTTGGTGGGACTGAACAC 36CAGAGAGCGCCTCCTATTCTAC 37 GGAGAGAGACCTGGGAAAAGTC 38AAGCGCTATGCAGAGAAGTACTC 39 GACACGTGCTTGAGGAAACAC 40GCAGACTAGGCTGCATTCACTC 41 AGAGCTGCGTGGACACTAC 42GGGAGAAAAAAGCCAACCTTAATGC 43 ATTAAACGATTACCCTGAGGATGATATGC 44GGTGCGTTTTCTTCCCATATTCC 45 TCTTTTATATCCCCTTCGTCTGCAAAC 46GTTAAATGGTGGTGGTGCATTCAC 47 GCGGTCCAAGGAATTTTGCTAAC 48CAAACACCCCTTATTCCAGTCAAAC 49 CCACTTTTTCCCAACCCCAAAATTC 50GCAGACCCAAGTTTCCTTTCTCC 51 CGGTTCCCACGAAAAGCAAC 52CACTGAGCAACACAATTGGACTTTC 53 CCTGGCTTATCACCTTGGACTTC 54TGTCTTGGGTGGGTTTCTCTTAAC 55 CCCAGACTCCTCCCTTGTTTC 56ACCTCAGCCATTGAACTCACTTC 57 CTGCTCTACTGGTTTCCCTAGATTC 58CCATAACCAGACTCAGCAGAGAAC 59 CAAACCCACCGCAGTAACTTAC 60GCCTGCCCAGGACTTACTC 61 AGGTATGTGGCTGCTGTCTTC 62 AAGGAGGCGTCGCTGTAC 63GCAGCCAGTCAACTCTGAAC 64 ACAGTCACCCTGCACACTTAC 65 CCCCGGGTGCGAAGTC 66

Example 15 Digital PCR Using APP

Digital PCR (dPCR) is a refinement of conventional polymerase chainreaction methods that can be used to directly quantify and clonallyamplify nucleic acids. With dPCR, a sample is partitioned so thatindividual nucleic acid molecules within the sample are localized andconcentrated within many separate regions. The partitioning of thesample allows one to count the molecules by estimating according toPoisson distribution. As a result, each part will contain “0” or “1”molecules, or a negative or positive reaction, respectively. After PCRamplification, nucleic acids may be quantified by counting the regionsthat contain PCR end-product, positive reactions. “The partitioning ofthe sample” can be accomplished in individual wells, such as wells in aplate, wells in a card, or micelles in an emulsion. Partition can alsobe done in space without real physical barrier, such as in a bridge PCR,“polony”, etc.

Conventionally, dPCR is widely monitored by TAQMAN detection. However,TAQMAN detection involves expensive probes that impede its widerapplication. This example demonstrates that APP, a cheaper approach, maybe ideal for this application and may be superior to regular dye-basedassay.

of 10 μL containing 50 mM Tris/HCl, pH8.5, 1.5 mM MgCl₂, 350 μMtetraphosphate, 0.5×SYBR Green, 31 μM dNTP, 15 mM (NH₃)₂SO₄, Tween 0.1%,6 ng/μl 10A, 500 nM PA14F and PA14R, 1.5 ng of human DNA. Each reactionwas cycled 50 times between 95° C. for 5 second and 65° C. for 1 minute.Of the 96 reactions, thirty-four (34) showed growth curves (FIG. 16A)and melting curves of Tm=86° C. (FIG. 16B). By definition, these arepositive reactions. The rest sixty-two (62) are negatives, exhibitingneither growth curves (FIG. 16C), nor melting curves (FIG. 16D).

When dPCR was conducted using regular dye-based PCR technologies, tofigure out positive from negatives were not straight forward.Specifically, regular dye-based dPCR was conducted in 96 partitions witheach partition of 10 μL containing 1×SYBR GreenER qPCR Supermix (LifeTechnologies Cat #11760-500), 500 nM PA14F, 500 nMPA14R, and 1.5 ng ofhuman DNA, in 50 cycles between 95° C. for 5 second and 65° C. for 1minute. All 96 partitions showed growth curves (FIGS. 17A and 17C). Notone reaction well showed a single melting curve peak at 86° C., as thecase in FIG. 16B. Instead, thirty nine (39) reaction wells have a peakat about 86° C. in addition to other peaks indicating additionalnon-specific amplification products. The other fifty seven (57) reactionwells also exhibited multiple peaks other than 86° C. The group ofthirty nine was assigned as positives, while the group of fifty sevenwas assigned as negatives. It is easier to discern the positives fromnegatives and bears higher confidence using APP-dPCR compared to regularPCR.

TABLE IV Primer Sequences Used in Digital PCR Primer NamePrimer Sequence SEQ ID Number vPA14F TCAGCACACCTATCGGAAAGCSEQ ID NO.: 68 vPA14R CCCAGACTCCTCCCTTGTTTC SEQ ID NO.: 69 PA14FTCAGCACACCTATCGGAAAG/ddC/ SEQ ID NO.: 68 PA14R CCCAGACTCCTCCCTTGTTT/ddC/SEQ ID NO.: 69

The distribution of Ct of negatives of APP, positives of APP, negativesof regular reaction and positives of regular reaction are plotted inFIG. 18A using JMP software. The same data was plotted in FIG. 18B toindicate the mean and 1× standard deviation. It is clear that indPCR-APP, the positives are well separated from the negatives based onCt, while in dPCR-regular, the positives cannot be separated from thenegatives based on Ct, demonstrating the utility and the superiority ofAPP.

The melting curves between FIG. 16B and FIG. 17B suggest that APP wouldgenerate much cleaner products in clonally amplify nucleic acids.

All references cited within this disclosure are hereby incorporated byreference in their entirety. While certain embodiments have beendescribed in terms of the preferred embodiments, it is understood thatvariations and modifications will occur to those skilled in the art.Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the followingclaims.

1-29. (canceled)
 30. A method for amplifying a target nucleic acid by anactivation by polyphosphorolysis (APP) reaction comprising the steps of:(a) annealing to a template strand of the target nucleic acid acomplementary activatable oligonucleotide P* having a non-extendablenucleotide at its 3′ terminus and having no mismatched nucleotides at ornear its 3′ terminus, so that the terminal nucleotide is hybridized tothe template strand when the oligonucleotide P* is annealed, (b)polyphosphorolyzing the resulting duplex with: 1) one or morepolyphosphorolyzing agents selected from the group consisting of acompound of the general formula I:

wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,8, 9, and 10; a compound of general formula II:

wherein n and/or m are selected from the group consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, and 10, with the proviso that n and m cannot both be0, and X is selected from the group consisting of:

 and 2) an enzyme having polyphosphorolysis activity that activates theoligonucleotide P* by removal of the hybridized terminal nucleotide, and(c) polymerizing a nucleic acid by extending the activatedoligonucleotide P* on the template strand in presence of four nucleosidetriphosphates and a nucleic acid polymerase to synthesize the desirednucleic acid strand; and (d) repeating the steps (a)-(c) as necessary toamplify the target nucleic acid.
 31. The method of claim 30 wherein theenzyme is a DNA polymerase.
 32. The method of claim 31 wherein theenzyme is a thermostable DNA polymerase.
 33. The method of claim 32wherein the thermostable DNA polymerase is selected from the groupconsisting of thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1,RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATORGAMMA.
 34. The method of claim 30 wherein the extending andamplification steps are carried out using a polymerase chain reaction.35. The method of claim 34 wherein the polymerase chain reaction is adigital polymerase chain reaction.
 36. The method of claim 30 using oneor more polyphosphorolyzing agents of Formula I:

wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,8, 9, and
 10. 37. The method of claim 30 using one or morepolyphosphorolyzing agents of Formula II:

where n and/or m are selected from the group consisting of 0, 1, 2, 3,4, 5, 6, 7, 8, 9, and 10, with the proviso that n and m cannot both be0, and X is selected from the group consisting of:


38. The method of claim 30 using one or more polyphosphorolyzing agentsselected from the group consisting of:


39. The method of claim 38 using one or more polyphosphorolyzing agentsselected from the group consisting of triphosphate, tetraphosphate, andimidodiphosphate.
 40. The method of claim 30, wherein thepolyphosphorolyzing further comprises using pyrophosphate in combinationwith the one or more polyphosphorolyzing agents.
 41. The method of claim30, further comprising detecting the polymerized nucleic acid.
 42. Themethod of claim 30, wherein the one or more polyphosphorolyzing agentsis present in the amplification reaction at a concentration ofapproximately 1-500 μM.
 43. The method of claim 30, wherein the targetnucleic acid is RNA.
 44. The method of claim 43 wherein the enzyme orpolymerase has reverse transcriptase activity.
 45. The method of claim43 wherein the enzyme is RQ1 or its mutant RQY.
 46. The method of claim43 used in a direct RNA sequencing reaction.